Brief Definitive Report
Upregulation and Coexpression of Adhesion Molecules Correlate with Relapsing Autoimmune Demyelination in the Central Nervous System By Barbara Cannella, Anne H . Cross, and Cedric S. Raine
From the Departments of Pathology (Neuropathology), Neurology, and Neuroscience, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, New York 10461
Summary The expression of adhesion molecules on central nervous system (CNS) vessels was examined during chronic relapsing experimental autoimmune encephalomyelitis in the SJL mouse. Two molecules associated with cell adhesion were studied : MECA 325, a murine lymph node high endothelial venule marker; and MALA2, the murine homologue of intercellular adhesion molecule 1. During initial disease, upregulated coexpression of these two molecules occurred in the CNS . This correlated with inflammatory cell invasion . During remission, expression was downregulated, and each subsequent relapse was accompanied by corresponding upregulation. Thus, up- and downregulation of adhesion molecules in the target organ appeared to form an integral part of the inflammatory process in this autoimmune condition and support a role for receptor-mediated inflammatory cell invasion of relevance to the pathogenesis of multiple sclerosis .
T
he mechanisms by which circulating immune cells recognize a target organ are not well understood, but in the case of the central nervous system (CNS), where the blood brain barrier (BBB) prevents free interchange between blood and CNS parenchyma, such mechanisms are probably fundamental to inflammation . In diseases of the CNS like multiple sclerosis (MS) and its laboratory model, experimental autoimmune encephalomyelitis (EAE), initiating events are believed to involve changes at the BBB resulting in increased vascular permeability. These events may facilitate the adhesion of lymphocytes to CNS endothelium, after which cells invade the target organ and initiate T cell-mediated demyelination (1) . Similar molecular events occur in lymph nodes, where, at the sites of lymphocyte extravasation, blood vessels have endothelial cells (EC) of a distinct cuboidal morphology : high endothelial venules (HEV) (2). In rheumatoid arthritis, EC acquire properties of HEV at sites of lymphocyte adhesion and invasion (3) . In adoptively transferred EAE, EC have been described with HEV morphology to which mononuclear cells were attached during lesion formation (4, 5). The present study has examined within the CNS during chronic relapsing adoptively transferred EAE, the expression of two molecules associated with cell adhesion: MECA-325, a murine HEV marker (6) ; and MALA2, the murine homologue of human intercellular adhesion molecule 1 (ICAM-1) (7, 8) . Materials and Methods
Animals and Immunization. Female SJL/J mice (The Jackson Laboratory, Bar Harbor, ME), 6-8 wks of age, were maintained 1521 J. Exp. Med. ® The Rockefeller Volume 172
November 1990
in an NIH/AAALAC approved facility. Donor SJL/J mice were immunized with guinea pig myelin basic protein (MBP) for the transfer of MBP-sensitized lymphocytes into naive, syngeneic recipients (9), and were sampled during the pre-clinical (24- 48 h before onset of signs), acute, remitting, and relapsing stages. Animals first showed signs of EAE 6-8 d post-transfer (PT) . Tissue Processing and Immunocytochemistry. Frozen sections (10 jam) of CNS tissue were fixed in acetone for 10 min and stained with the avidin-biotin-peroxidase complex (ABC) technique and 3',3-diaminobenzidine as the chromagen . As controls, kidney and lymph node tissues were studied. Incubations were performed overnight with the rat monoclonals MECA 325 (6), used at a 1 :10 dilution ; YNI/1, which detects MALA2 (7, 8), used as straight supernate ; anti-lymphocyte function-associated antigen (LFA-1) (Boehringer-Mannheim Biochemicals, Indianapolis, IN), used at 1 :20; biotinylated la' (from MKS-4 hybridoma) (American Type Culture Collection, Rockville, MD), purified by affinity chromatography, used at 1 :25; and anti-L3T4 (Becton Dickinson & Co., Mountain View, CA), used at 1:100. Control sections were incubated with PBS. For double labeling, rabbit anti-glial fibrillary acidic protein (GFAP) antibody, (gift of J.E. Goldman, Columbia University), was used at 1:100. Results Spinal cord sections reacted for MECA-325 showed that during the preclinical phase, this HEV marker was expressed at low levels on the surface of EC. It was not seen in the CNS of normal mice. MECA325 expression was greatly upregulated on the majority of postcapillary venules in spinal cord white matter lesions during the acute phase within 24University Press " 0022-1007/90/11/1521/04 $2 .00 1521-1524
48 h after onset of paralysis, at which timepoint inflammatory cell invasion was prominent (Fig. 1 a). Unaffected white matter, grey matter, and rostral CNS regions showed no MECA-325 reactivity. MECA-325 decreased during clinical remissions to near preclinical levels (Fig. 1 b), and then increased markedly again during each relapse (Fig. 1 c) . Differences in MECA-325 staining between preclinical and acute
stages, and between relapses and remissions, were statistically significant (Fig . 2) . Low levels of ICAM-1 were noted in the spinal cord during the preclinical phase, similar to MECA-325 . ICAM-1 was greatly upregulated in lesion areas during the acute phase (Fig. 1 d), tapering off during remission (Fig. 1 e). ICAM-1 expression subsequently increased with each relapse (Fig. 1 f),
Figure 1 . Sections of spinal cord tissue from mice with chronic relapsing EAE demonstrate fluctuations in expression of adhesion molecules, MECA325 and MALA-2 (ICAM-1), coincident with clinical activity. Upregulated expression of MECA-325 is seen on the surface of EC during the acute phase of adoptively transferred EAE (a) ; this decreases during clinical remission (b), and increases again during relapse (c) . The same fluctuating pattern is shown for MALA-2 expression during acute disease (d), remissions (e), and relapses (f) . In e, note the lack of expression along the majority of the vessels, the small area to the right which is MALA-2 * , and the positively staining astrocytes (armws) . a-f (x550). 1522
Adhesion Molecule Expression during Demyelination
Figure 2 . A histogram illustrates the up- and downregulation of adhesion molecules in the CNS of mice with adoptively transferred EAE correlating with clinical activity. Expression of MECA-325, MALA-2, and LFA-1 was significantly different when the preclinical vs. acute stage, and relapses vs . remissions were compared. MECA-325, p < 0.002 and p < 0 .05; MALA-2, p < 0.05 and p < 0.01 ; LFA-1, p < 0 .05 and p < 0 .01, respectively. Differences in la expression were significant only for preclinical vs. acute stages (p < 0.05), and in CD4 expression, for relapses vs. remissions (p < 0.05) . The score was based on the density of positive immunoperoxidase-labeled cells as determined by two blinded observers. Frozen sections from 15 animals were analyzed . Numerical values (0-4) were assigned corresponding to the percentage scale, and significance was determined by the student's t test .
and was found on EC of all vessels, perivascular cells, and scattered astrocytes (Fig. 1 e), which were also GFAP+ . Unlike MECA-325, low levels were seen on EC and glial cells in the CNS of normal mice. Statistical comparison of ICAM-1 at various stages of disease revealed the differences to be significant (Fig . 2) . Infiltration of the CNS correlated with clinical onset, as has been demonstrated previously (9) . The majority of the infiltrating cells were LFA1' . LFA1 expression also closely paralleled that of its ligand, ICAM-1, and of MECA-325 (Fig. 2) . Class 11 MHC molecules, also implicated in adhesion (10, 11) were expressed during the acute and relapsing phases, similar to MECA325 and ICAM-1 (Fig. 2). MHC class II expression occurred on perivascular cells ; CNS EC appeared to be MHC class II negative . T cells expressing CD4, the ligand for MHC class II, were first demonstrated in the CNS during the preclinical stage and increased during the acute and relapsing phases . During remissions, CD4' cells decreased in number. To test whether the fluctuation in adhesion molecule expression was target organ specific, kidney sections from each group (preclinical, acute, remission, and relapse) were tested for MECA325, ICAM-1, and MHC class 11, and were uniformly negative. As a positive control, lymph node tissue was routinely included and was always positive for MECA-325, ICAM-1, LFA1, MHC class II, and CD4 . Discussion The coexpression of two distinct adhesion-associated molecules not normally seen in CNS tissue has been shown within 1523
Cannella et al.
the CNS of mice with relapsing autoimmune demyelination. One of these, MECA325, is normally present only in lymph node tissue, and the other, ICAM-1, while most common in hematopoietic tissue, is seen at low levels in several organs, including the CNS (12). Our study has shown a close correlation of disease activity with adhesion molecule expression. Since understanding of the mechanisms in the trafficking of immune cells to CNS white matter might be fundamental to the inflammatory demyelinating diseases, we compared the expression of a number of adhesion-related molecules within the CNS at different stages of chronic relapsing EAE . The appearance of MECA-325 occurred at the'BBB and paralleled lymphocytic infiltration . To our knowledge, this represents the first demonstration of an HEV determinant at CNS inflammatory sites . Whether the selective induction of this HEV molecule preceded or occurred during lymphocyte extravasation is not yet clear. The data suggest that the differentiation of HEVlike vessels was a gradual process occurring simultaneously with increasing lymphocytic influx . The present results support the concept of cell traffic being associated with the modulation of expression of HEV molecules on target organ vessels. The reasons for these fluctuations remain speculative but may relate to cytokines elaborated locally. For example, interferon y, a product of activated T cells, is known to induce MECA-325, ICAM-1, and MHC class II on a variety of cells, including EC (6, 13, 14) . Previous studies using 14C-labeled T cell blasts to induce adoptively transferred EAE showed that some MBP-immune cells arrived at the CNS vasculature 24-48 h before clinical onset (4) . The arrival of radiolabeled cells appeared to precede unlabeled cells, suggesting that the original CNS-immune cells induced local changes at the BBB, which then precipitated the influx of CNS antigen-nonspecific cells (4, 5). These events may have morphologic correlates in that by ultrastructure, HEVlike vessels with unique membrane specializations occurred in the CNS during early EAE (5) . MECA-325 is a member of the addressin family, a group of molecules whose apparent role is to signal and direct cellto-cell interactions. Its ligand is unknown . With regard to function, MECA325 antibodies do not inhibit lymphocyte binding, thus not supporting its direct role in cell adhesion (15). MECA-325 may define a distinct epitope on HEV involved in some other aspect of lymphocyte/endothelial cell interactions (15) . The inappropriate presence of HEVlike vessels in the CNS, coupled with the fluctuations of MECA 325 with disease activity, appear to implicate this molecule in EAE. Parallel studies using an antibody against a constitutive antigen, von Willebrandt Factor (vWF), confirmed that the MECA-325 reactivity was specific. Cell adhesion is an essential component of the response of T lymphocytes to antigens. Our demonstration of increased ICAM-1 in the CNS, particularly on EC, is the first to support a role for ICAM-1 during inflammation and demyelination in EAE. ICAM-1 is a member of the Ig gene superfamily and has been associated with activated lymphocytes and sites of inflammation (13). The present fluctuations in ICAM-1 expression and its receptor LFA1 (16, 17), on inflammatory cells, suggest this interaction may play a role in the
Brief Definitive Report
mediation of lymphocytic traffic into CNS inflammatory sites. This speculation is supported by a recent study showing ICAM-1 in human MS and control CNS lesions (12) . The expression of MECA 325, ICAM-1, and MHC class II appears to be important in leukocyte homing and adhesion to the BBB during active stages of autoimmune demyelination . Their fluctuating patterns of expression corresponding to periods of clinical worsening support a functional role .
Non-CNS tissue from the same animals demonstrated no expression of any of the above molecules, indicating their expression to be target organ related. Since several adhesion molecules appear to be coexpressed and function cooperatively in lymphocyte homing to the CNS, the molecular events regulating cellular migration to the target organ during chronic relapsing EAE appear to be synchronized and multifactorial.
We thank Drs. A. M. Duijvestijn (University of Limburg, Maastricht, The Netherlands) for providing MECA-325 ; K. Horley and F. Takei (University of British Columbia, Vancouver, Canada) for providing anti-MA-LA-2 ; Dr. C. F. Brosnan for helpful discussions ; M. Briggs for preparing the manuscript ; and E. Swanson, M. Pakingan, and H. Finch for technical assistance . This work was supported by the National Institutes of Health (NS-11920, NS-08952, NS-07098); and the National Multiple Sclerosis Society (RG-1001-G-7 ; FG-749-A-1) . Address correspondence to Barbara Cannella, Department of Pathology (Neuropathology) K435, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
Received for publication 9 July 1990 and in revised form 29 August 1990 .
References
1 . Paterson, P.Y. 1977 . Autoimmune neurological diseases : experimental animal systems and implications for multiple sclerosis. In Autoimmunity: Genetic, Immunologic, Virologic, and Clinical Aspects. N. Talal, editor. Academic Press, New York . 643-692 . 2. Gowans, J.L ., and E.J . Knight . 1964 . The route of recirculation of lymphocytes in the rat . Proc R. Soc. Lond. B Biol. Sci. 159:257 . 3. Jalkanen, S., A.C. Steere, R.I . Fox, and E.C . Butcher. 1986 . A distinct endothelial cell recognition system that controls lymphocytic traffic into inflamed synovium . Science (Wash. DC). 233:556 . 4. Cross, A.H ., B. Cannella, C.F. Brosnan, and C.S. Raine. 1990 . Homing to central nervous system vasculature by antigen specific lymphocytes . I. Localization of "C-labeled cells during acute, chronic and relapsing experimental allergic encephalomyelitis. Lah Invest. 63 :162 . 5. Raine, C.S., B. Cannella, A.M . Duijvestijn, and A.H . Cross. 1990. Homing to central nervous system vasculature by antigen specific lymphocytes. II. Lymphocyte/endothelial cell adhesion during the initial stages of autoimmune demyelination . Lah Invest. In press. 6. Duijvestijn, A.M ., A.B. Schreiber, and E.C . Butcher. 1986 . Interferon-gamma regulates an antigen specific for endothelial cells involved in lymphocyte traffic. Proc Nad. Acad. Sci. USA . 83 :9114. 7. Takei, F. 1985 . Inhibition of mixed lymphocyte response by a rat monoclonal antibody to a novel murine lymphocyte activation antigen (MALA2). J. Immunol. 134:1403. 8. Horley, KJ ., C. Carpenito, B. Baker, and F. Takei. 1989 . Molecular cloning of murine intercellular adhesion molecule (ICAM-1) . EMBO (Eur. Mot. BiA Organ) J. 8:2880. 9. Mokhtarian, F., D.E . McFarlin, and C.S . Raine. 1984 . Adop1524
10 . 11 . 12 . 13 .
14 .
15 .
16 .
17 .
tive transfer o£ myelin basic protein-sensitized T cells produces chronic relapsing demyelinating disease in mice. Nature (Land.). 309:356 . Masuyama, J., N . Minato, and S. Kano. 1986 . Mechanisms of lymphocyte adhesion to human vascular endothelial cells in culture. J. Clin . Invest. 77 :1596. Doyle, C., andJ.L . Strominger. 1987 . Interactio n between CD4 and class II MHC molecules mediates cell adhesion . Nature (Lon4 330:256 . Sobel, R., M.E . Mitchell, and G. Fondren. 1990 . Intercellular adhesion molecule-1 (ICAM-1) in cellular immune reactions in the human central nervous system. Am . J. Path. 136:1309. Dustin, M.L ., F. Rothlein, A.K . Bhan, C.A . Dinarello, and R.A . Springer. 1986 . Induction by IL1 and interferon-gamma : tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-1) . J. Immunol. 137:245 . Pober, J.S., M.A . Gimbrone, R.S . Cotran, C.S . Reiss, S.J . Burakoff, WFiers, and K.A. Ault . 1983 . la expression by vascular endothelium is inducible by activated T cells and by human .y interferon . J. Exp Med. 157:1339. Duijvestijn, A.M ., M. Kerkhove, R.F. Bargatze, and E .C . Butcher. 1987 . Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies . J. Immunol. 138:713 . Springer, R.A., M.L . Dusin, T.K . Kishimoto, and S.D. Marlin . 1987. The lymphocyte function associated LFA1, CD2, and LFA3 molecules. Cell adhesion receptors of the immune system . Annu . Rev. Immunol. 5 :223 . Hammann, A., D. JablonskiWestrich, A. Duijvestijn, E.C . Butcher, H. Baisch, T Harder, and H.G. Thiele. 1988 . Evidence for accessory role of LFA1 in lymphocyte-high endothelium interaction during homing. J. Immunol. 140:693 .
Adhesion Molecule Expression during Demyelination
Upregulation and Coexpression of Adhesion Molecules Correlate with Relapsing Autoimmune Demyelination in the Central Nervous System By Barbara Cannella, Anne H . Cross, and Cedric S. Raine
From the Departments of Pathology (Neuropathology), Neurology, and Neuroscience, and the Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, New York 10461
Summary The expression of adhesion molecules on central nervous system (CNS) vessels was examined during chronic relapsing experimental autoimmune encephalomyelitis in the SJL mouse. Two molecules associated with cell adhesion were studied : MECA 325, a murine lymph node high endothelial venule marker; and MALA2, the murine homologue of intercellular adhesion molecule 1. During initial disease, upregulated coexpression of these two molecules occurred in the CNS . This correlated with inflammatory cell invasion . During remission, expression was downregulated, and each subsequent relapse was accompanied by corresponding upregulation. Thus, up- and downregulation of adhesion molecules in the target organ appeared to form an integral part of the inflammatory process in this autoimmune condition and support a role for receptor-mediated inflammatory cell invasion of relevance to the pathogenesis of multiple sclerosis .
T
he mechanisms by which circulating immune cells recognize a target organ are not well understood, but in the case of the central nervous system (CNS), where the blood brain barrier (BBB) prevents free interchange between blood and CNS parenchyma, such mechanisms are probably fundamental to inflammation . In diseases of the CNS like multiple sclerosis (MS) and its laboratory model, experimental autoimmune encephalomyelitis (EAE), initiating events are believed to involve changes at the BBB resulting in increased vascular permeability. These events may facilitate the adhesion of lymphocytes to CNS endothelium, after which cells invade the target organ and initiate T cell-mediated demyelination (1) . Similar molecular events occur in lymph nodes, where, at the sites of lymphocyte extravasation, blood vessels have endothelial cells (EC) of a distinct cuboidal morphology : high endothelial venules (HEV) (2). In rheumatoid arthritis, EC acquire properties of HEV at sites of lymphocyte adhesion and invasion (3) . In adoptively transferred EAE, EC have been described with HEV morphology to which mononuclear cells were attached during lesion formation (4, 5). The present study has examined within the CNS during chronic relapsing adoptively transferred EAE, the expression of two molecules associated with cell adhesion: MECA-325, a murine HEV marker (6) ; and MALA2, the murine homologue of human intercellular adhesion molecule 1 (ICAM-1) (7, 8) . Materials and Methods
Animals and Immunization. Female SJL/J mice (The Jackson Laboratory, Bar Harbor, ME), 6-8 wks of age, were maintained 1521 J. Exp. Med. ® The Rockefeller Volume 172
November 1990
in an NIH/AAALAC approved facility. Donor SJL/J mice were immunized with guinea pig myelin basic protein (MBP) for the transfer of MBP-sensitized lymphocytes into naive, syngeneic recipients (9), and were sampled during the pre-clinical (24- 48 h before onset of signs), acute, remitting, and relapsing stages. Animals first showed signs of EAE 6-8 d post-transfer (PT) . Tissue Processing and Immunocytochemistry. Frozen sections (10 jam) of CNS tissue were fixed in acetone for 10 min and stained with the avidin-biotin-peroxidase complex (ABC) technique and 3',3-diaminobenzidine as the chromagen . As controls, kidney and lymph node tissues were studied. Incubations were performed overnight with the rat monoclonals MECA 325 (6), used at a 1 :10 dilution ; YNI/1, which detects MALA2 (7, 8), used as straight supernate ; anti-lymphocyte function-associated antigen (LFA-1) (Boehringer-Mannheim Biochemicals, Indianapolis, IN), used at 1 :20; biotinylated la' (from MKS-4 hybridoma) (American Type Culture Collection, Rockville, MD), purified by affinity chromatography, used at 1 :25; and anti-L3T4 (Becton Dickinson & Co., Mountain View, CA), used at 1:100. Control sections were incubated with PBS. For double labeling, rabbit anti-glial fibrillary acidic protein (GFAP) antibody, (gift of J.E. Goldman, Columbia University), was used at 1:100. Results Spinal cord sections reacted for MECA-325 showed that during the preclinical phase, this HEV marker was expressed at low levels on the surface of EC. It was not seen in the CNS of normal mice. MECA325 expression was greatly upregulated on the majority of postcapillary venules in spinal cord white matter lesions during the acute phase within 24University Press " 0022-1007/90/11/1521/04 $2 .00 1521-1524
48 h after onset of paralysis, at which timepoint inflammatory cell invasion was prominent (Fig. 1 a). Unaffected white matter, grey matter, and rostral CNS regions showed no MECA-325 reactivity. MECA-325 decreased during clinical remissions to near preclinical levels (Fig. 1 b), and then increased markedly again during each relapse (Fig. 1 c) . Differences in MECA-325 staining between preclinical and acute
stages, and between relapses and remissions, were statistically significant (Fig . 2) . Low levels of ICAM-1 were noted in the spinal cord during the preclinical phase, similar to MECA-325 . ICAM-1 was greatly upregulated in lesion areas during the acute phase (Fig. 1 d), tapering off during remission (Fig. 1 e). ICAM-1 expression subsequently increased with each relapse (Fig. 1 f),
Figure 1 . Sections of spinal cord tissue from mice with chronic relapsing EAE demonstrate fluctuations in expression of adhesion molecules, MECA325 and MALA-2 (ICAM-1), coincident with clinical activity. Upregulated expression of MECA-325 is seen on the surface of EC during the acute phase of adoptively transferred EAE (a) ; this decreases during clinical remission (b), and increases again during relapse (c) . The same fluctuating pattern is shown for MALA-2 expression during acute disease (d), remissions (e), and relapses (f) . In e, note the lack of expression along the majority of the vessels, the small area to the right which is MALA-2 * , and the positively staining astrocytes (armws) . a-f (x550). 1522
Adhesion Molecule Expression during Demyelination
Figure 2 . A histogram illustrates the up- and downregulation of adhesion molecules in the CNS of mice with adoptively transferred EAE correlating with clinical activity. Expression of MECA-325, MALA-2, and LFA-1 was significantly different when the preclinical vs. acute stage, and relapses vs . remissions were compared. MECA-325, p < 0.002 and p < 0 .05; MALA-2, p < 0.05 and p < 0.01 ; LFA-1, p < 0 .05 and p < 0 .01, respectively. Differences in la expression were significant only for preclinical vs. acute stages (p < 0.05), and in CD4 expression, for relapses vs. remissions (p < 0.05) . The score was based on the density of positive immunoperoxidase-labeled cells as determined by two blinded observers. Frozen sections from 15 animals were analyzed . Numerical values (0-4) were assigned corresponding to the percentage scale, and significance was determined by the student's t test .
and was found on EC of all vessels, perivascular cells, and scattered astrocytes (Fig. 1 e), which were also GFAP+ . Unlike MECA-325, low levels were seen on EC and glial cells in the CNS of normal mice. Statistical comparison of ICAM-1 at various stages of disease revealed the differences to be significant (Fig . 2) . Infiltration of the CNS correlated with clinical onset, as has been demonstrated previously (9) . The majority of the infiltrating cells were LFA1' . LFA1 expression also closely paralleled that of its ligand, ICAM-1, and of MECA-325 (Fig. 2) . Class 11 MHC molecules, also implicated in adhesion (10, 11) were expressed during the acute and relapsing phases, similar to MECA325 and ICAM-1 (Fig. 2). MHC class II expression occurred on perivascular cells ; CNS EC appeared to be MHC class II negative . T cells expressing CD4, the ligand for MHC class II, were first demonstrated in the CNS during the preclinical stage and increased during the acute and relapsing phases . During remissions, CD4' cells decreased in number. To test whether the fluctuation in adhesion molecule expression was target organ specific, kidney sections from each group (preclinical, acute, remission, and relapse) were tested for MECA325, ICAM-1, and MHC class 11, and were uniformly negative. As a positive control, lymph node tissue was routinely included and was always positive for MECA-325, ICAM-1, LFA1, MHC class II, and CD4 . Discussion The coexpression of two distinct adhesion-associated molecules not normally seen in CNS tissue has been shown within 1523
Cannella et al.
the CNS of mice with relapsing autoimmune demyelination. One of these, MECA325, is normally present only in lymph node tissue, and the other, ICAM-1, while most common in hematopoietic tissue, is seen at low levels in several organs, including the CNS (12). Our study has shown a close correlation of disease activity with adhesion molecule expression. Since understanding of the mechanisms in the trafficking of immune cells to CNS white matter might be fundamental to the inflammatory demyelinating diseases, we compared the expression of a number of adhesion-related molecules within the CNS at different stages of chronic relapsing EAE . The appearance of MECA-325 occurred at the'BBB and paralleled lymphocytic infiltration . To our knowledge, this represents the first demonstration of an HEV determinant at CNS inflammatory sites . Whether the selective induction of this HEV molecule preceded or occurred during lymphocyte extravasation is not yet clear. The data suggest that the differentiation of HEVlike vessels was a gradual process occurring simultaneously with increasing lymphocytic influx . The present results support the concept of cell traffic being associated with the modulation of expression of HEV molecules on target organ vessels. The reasons for these fluctuations remain speculative but may relate to cytokines elaborated locally. For example, interferon y, a product of activated T cells, is known to induce MECA-325, ICAM-1, and MHC class II on a variety of cells, including EC (6, 13, 14) . Previous studies using 14C-labeled T cell blasts to induce adoptively transferred EAE showed that some MBP-immune cells arrived at the CNS vasculature 24-48 h before clinical onset (4) . The arrival of radiolabeled cells appeared to precede unlabeled cells, suggesting that the original CNS-immune cells induced local changes at the BBB, which then precipitated the influx of CNS antigen-nonspecific cells (4, 5). These events may have morphologic correlates in that by ultrastructure, HEVlike vessels with unique membrane specializations occurred in the CNS during early EAE (5) . MECA-325 is a member of the addressin family, a group of molecules whose apparent role is to signal and direct cellto-cell interactions. Its ligand is unknown . With regard to function, MECA325 antibodies do not inhibit lymphocyte binding, thus not supporting its direct role in cell adhesion (15). MECA-325 may define a distinct epitope on HEV involved in some other aspect of lymphocyte/endothelial cell interactions (15) . The inappropriate presence of HEVlike vessels in the CNS, coupled with the fluctuations of MECA 325 with disease activity, appear to implicate this molecule in EAE. Parallel studies using an antibody against a constitutive antigen, von Willebrandt Factor (vWF), confirmed that the MECA-325 reactivity was specific. Cell adhesion is an essential component of the response of T lymphocytes to antigens. Our demonstration of increased ICAM-1 in the CNS, particularly on EC, is the first to support a role for ICAM-1 during inflammation and demyelination in EAE. ICAM-1 is a member of the Ig gene superfamily and has been associated with activated lymphocytes and sites of inflammation (13). The present fluctuations in ICAM-1 expression and its receptor LFA1 (16, 17), on inflammatory cells, suggest this interaction may play a role in the
Brief Definitive Report
mediation of lymphocytic traffic into CNS inflammatory sites. This speculation is supported by a recent study showing ICAM-1 in human MS and control CNS lesions (12) . The expression of MECA 325, ICAM-1, and MHC class II appears to be important in leukocyte homing and adhesion to the BBB during active stages of autoimmune demyelination . Their fluctuating patterns of expression corresponding to periods of clinical worsening support a functional role .
Non-CNS tissue from the same animals demonstrated no expression of any of the above molecules, indicating their expression to be target organ related. Since several adhesion molecules appear to be coexpressed and function cooperatively in lymphocyte homing to the CNS, the molecular events regulating cellular migration to the target organ during chronic relapsing EAE appear to be synchronized and multifactorial.
We thank Drs. A. M. Duijvestijn (University of Limburg, Maastricht, The Netherlands) for providing MECA-325 ; K. Horley and F. Takei (University of British Columbia, Vancouver, Canada) for providing anti-MA-LA-2 ; Dr. C. F. Brosnan for helpful discussions ; M. Briggs for preparing the manuscript ; and E. Swanson, M. Pakingan, and H. Finch for technical assistance . This work was supported by the National Institutes of Health (NS-11920, NS-08952, NS-07098); and the National Multiple Sclerosis Society (RG-1001-G-7 ; FG-749-A-1) . Address correspondence to Barbara Cannella, Department of Pathology (Neuropathology) K435, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
Received for publication 9 July 1990 and in revised form 29 August 1990 .
References
1 . Paterson, P.Y. 1977 . Autoimmune neurological diseases : experimental animal systems and implications for multiple sclerosis. In Autoimmunity: Genetic, Immunologic, Virologic, and Clinical Aspects. N. Talal, editor. Academic Press, New York . 643-692 . 2. Gowans, J.L ., and E.J . Knight . 1964 . The route of recirculation of lymphocytes in the rat . Proc R. Soc. Lond. B Biol. Sci. 159:257 . 3. Jalkanen, S., A.C. Steere, R.I . Fox, and E.C . Butcher. 1986 . A distinct endothelial cell recognition system that controls lymphocytic traffic into inflamed synovium . Science (Wash. DC). 233:556 . 4. Cross, A.H ., B. Cannella, C.F. Brosnan, and C.S. Raine. 1990 . Homing to central nervous system vasculature by antigen specific lymphocytes . I. Localization of "C-labeled cells during acute, chronic and relapsing experimental allergic encephalomyelitis. Lah Invest. 63 :162 . 5. Raine, C.S., B. Cannella, A.M . Duijvestijn, and A.H . Cross. 1990. Homing to central nervous system vasculature by antigen specific lymphocytes. II. Lymphocyte/endothelial cell adhesion during the initial stages of autoimmune demyelination . Lah Invest. In press. 6. Duijvestijn, A.M ., A.B. Schreiber, and E.C . Butcher. 1986 . Interferon-gamma regulates an antigen specific for endothelial cells involved in lymphocyte traffic. Proc Nad. Acad. Sci. USA . 83 :9114. 7. Takei, F. 1985 . Inhibition of mixed lymphocyte response by a rat monoclonal antibody to a novel murine lymphocyte activation antigen (MALA2). J. Immunol. 134:1403. 8. Horley, KJ ., C. Carpenito, B. Baker, and F. Takei. 1989 . Molecular cloning of murine intercellular adhesion molecule (ICAM-1) . EMBO (Eur. Mot. BiA Organ) J. 8:2880. 9. Mokhtarian, F., D.E . McFarlin, and C.S . Raine. 1984 . Adop1524
10 . 11 . 12 . 13 .
14 .
15 .
16 .
17 .
tive transfer o£ myelin basic protein-sensitized T cells produces chronic relapsing demyelinating disease in mice. Nature (Land.). 309:356 . Masuyama, J., N . Minato, and S. Kano. 1986 . Mechanisms of lymphocyte adhesion to human vascular endothelial cells in culture. J. Clin . Invest. 77 :1596. Doyle, C., andJ.L . Strominger. 1987 . Interactio n between CD4 and class II MHC molecules mediates cell adhesion . Nature (Lon4 330:256 . Sobel, R., M.E . Mitchell, and G. Fondren. 1990 . Intercellular adhesion molecule-1 (ICAM-1) in cellular immune reactions in the human central nervous system. Am . J. Path. 136:1309. Dustin, M.L ., F. Rothlein, A.K . Bhan, C.A . Dinarello, and R.A . Springer. 1986 . Induction by IL1 and interferon-gamma : tissue distribution, biochemistry and function of a natural adherence molecule (ICAM-1) . J. Immunol. 137:245 . Pober, J.S., M.A . Gimbrone, R.S . Cotran, C.S . Reiss, S.J . Burakoff, WFiers, and K.A. Ault . 1983 . la expression by vascular endothelium is inducible by activated T cells and by human .y interferon . J. Exp Med. 157:1339. Duijvestijn, A.M ., M. Kerkhove, R.F. Bargatze, and E .C . Butcher. 1987 . Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies . J. Immunol. 138:713 . Springer, R.A., M.L . Dusin, T.K . Kishimoto, and S.D. Marlin . 1987. The lymphocyte function associated LFA1, CD2, and LFA3 molecules. Cell adhesion receptors of the immune system . Annu . Rev. Immunol. 5 :223 . Hammann, A., D. JablonskiWestrich, A. Duijvestijn, E.C . Butcher, H. Baisch, T Harder, and H.G. Thiele. 1988 . Evidence for accessory role of LFA1 in lymphocyte-high endothelium interaction during homing. J. Immunol. 140:693 .
Adhesion Molecule Expression during Demyelination
